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TITLE: Non-selective Persistence of a Rickettsia conorii Extrachromosomal Plasmid 1

During Mammalian Infection 2

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RUNNING TITLE: Rickettsia plasmids in mammalian infection 4

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AUTHORS: Sean P Riley1,2, Abigail I Fish1,2, Daniel A Garza1,2, Kaikhushroo H 6

Banajee1,2, Emma K Harris1,2, Fabio del Piero2, Juan J Martinez1,2,# 7

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AFFILIATIONS: 1 Vector Borne Disease Laboratories, 2 Department of Pathobiological 9

Sciences, Louisiana State University School of Veterinary Medicine, Baton Rouge, LA 10

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# CORRESPONDING AUTHOR: [email protected], (225) 578-929712

IAI Accepted Manuscript Posted Online 11 January 2016Infect. Immun. doi:10.1128/IAI.01205-15Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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ABSTRACT: 13

Scientific analysis of the genus Rickettsia is undergoing a rapid period of change with 14

the emergence of viable genetic tools. The development of these tools for mutagenesis 15

of pathogenic bacteria will permit forward genetic analysis of Rickettsia pathogenesis. 16

Despite these advances, uncertainty still remains regarding the use of plasmids in 17

studying these bacteria during in vivo mammalian models of infection; namely the 18

potential for virulence changes associated with presence of extrachromosomal DNA 19

and non-selective persistence of plasmids during mammalian models of infection. 20

Herein, we describe transformation of Rickettsia conorii Malish 7 with the plasmid 21

pRam18dRGA[AmTrCh]. Transformed R. conorii stably maintains this plasmid in 22

infected cell cultures, expresses the encoded fluorescent proteins, and exhibits growth 23

kinetics in cell culture similar to non-transformed R. conorii. Using a well-established 24

fatal murine model of Mediterranean spotted fever, we demonstrate that R. conorii 25

(pRam18dRGA[AmTrCh]) elicit the same fatal outcomes in animals as the 26

untransformed counterparts and ,importantly, maintain the plasmid throughout the 27

infection in the absence of selective antibiotic pressure. Interestingly, plasmid-28

transformed R. conorii were readily observed both in endothelial cells and also within 29

circulating leukocytes. Together, our data demonstrate that the presence of an 30

extrachromosomal DNA element in a pathogenic rickettsial species does not affect 31

either in vitro proliferation or in vivo infectivity in models of disease, and that plasmids 32

such as pRam18dRGA[AmTrCh] are valuable tools for further genetic manipulation of 33

pathogenic Rickettsia.34


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INTRODUCTION: 35

Rickettsia conorii is a pathogenic member of the spotted fever group (SFG) 36

Rickettsia. Multiple tick-borne SFG Rickettsia species can cause a wide range of 37

human and zoonotic diseases, with untreated cases of Rocky Mountain spotted fever 38

(R. rickettsii) disease resulting in 20-25% mortality (1-3). The similar disease in 39

Eurasia, Mediterranean spotted fever (MSF), is caused by R. conorii and results in 2-40

32% mortality in reported human cases (4). Symptoms from these diseases manifest 2-41

14 days following arthropod transmission, and are the result of widespread bacterial 42

infection of the endothelium with concurrent inflammation (5, 6). While broad-spectrum 43

antibiotic treatment can significantly reduce morbidity and mortality, untreated infections 44

can proceed to the more severe disease manifestations, including pulmonary edema, 45

interstitial pneumonia, and multi-organ failure (7). 46

Historically, analysis of the genetic determinants of SFG Rickettsia disease has 47

been hampered by a lack of viable tools for genetic manipulation of these bacteria. This 48

genetic recalcitrance was largely a consequence of lack of both anexic growth medium 49

and selectable markers. However, the development of tools for chromosomal 50

mutagenesis and selectively-maintained extrachromosomal DNA in rickettsial species 51

has been demonstrated (8). Plasmids isolated from non-pathogenic Rickettsia have 52

been adapted for replication and selection in both Rickettsia and Escherichia species 53

(9). These plasmids can potentially be useful for heterologous gene expression, 54

reporter gene expression, and in trans complementation in diverse Rickettsia species. 55

One such plasmid, pRam18dRGA[AmTrCh], is derived from the 18kbp plasmid 56

from R. amblyommii (10, 11). The nonessential portion of the naturally occurring 57


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plasmid was removed, and the remaining portion was inserted into an E. coli vector. 58

The gfpuv/rpaar2 genes, and a multiple cloning site (MCS) were introduced to produce 59

the plasmid pRam18dRGA[MCS] (9, 12, 13). The 948bp AmTrCh cassette encoding for 60

mCherry was subsequently added to create pRam18dRGA[AmTrCh] (9, 14). This 61

plasmid has been successfully transformed into five pathogenic and non-pathogenic 62

Rickettsia species (9, 15). 63

Herein, we utilize this plasmid for transformation of pathogenic R. conorii to 64

examine the in vivo persistence of an extrachromosomal DNA element in the absence 65

of antibiotic selection, and to determine whether the presence of the plasmid affects the 66

virulence of transformed R. conorii Malish 7 in a murine model of MSF. These analyses 67

subsequently yielded identification of new host cell types that are parasitized by R. 68

conorii in vivo.69


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MATERIALS AND METHODS: 70

Bacterial transformation. R. conorii strain Malish 7 was electroporated with 71

pRam18dRGA[AmTrCh] in a manner similar to Rackek, et.al (16). Briefly, 72

approximately 4x108 R. conorii were purified by sucrose density centrifugation (17). 73

The bacteria were made electro-competent through washing 4 times with ice-cold 74

250mM sucrose. 10µL of 1mg/mL pRam18dRGA[AmTrCh] was added to 4x107 75

electrocompetent R. conorii in a 0.1cm-gap electroporation cuvette. After 30 minutes of 76

incubation on ice, the sample was electroporated at 1700V, 150Ω, 50µF with a resulting 77

time constant of approximately 6.5ms. This mixture was immediately transferred to 1mL 78

of 8x106 Vero cells in Hank’s Balanced Salt Solution. After 1 hour of incubation at 34°C, 79

5% CO2 with rotation, the mixture was transferred to a 75cm flask containing 15mL 80

DMEM+10% BSA+ 1% Non-essential Amino Acids. After 24 hours of incubation at 81

34ºC 5% CO2 the media was changed to complete DMEM with 200ng/mL Rifampicin. 82

The media was replaced every 3 days until bacilliary growth was observed by Diff-Quik 83

staining. 84

Sorting and recovery of transformants. Single bacterium cell sorting was performed 85

as described in (18). Briefly, the PerCP/Cy5.5 fluorochrome was conjugated to the anti-86

R. conorii antibody RcPFA (19) using the Lightning-Link PerCP/Cy5.5 conjugation kit 87

(Innova Biosciences). R. conorii (pRam18dRGA[AmTrCh]) were stained with anti-R. 88

conorii antibody RcPFA-PerCP/Cy5.5 at 10 µg/mL. Labeled R. conorii were applied to a 89

FACSJazz cell sorter with forward scatter (FSC) threshold/trigger, side scatter (SSC) 90

versus FSC graph, and SSC versus log 488nm excitation 692/30nm emission graph. 91

Lysed Vero cells and non-transformed R. conorii were used as controls to set negative 92


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and positive sort gates. pRam18dRGA[AmTrCh] transformed R. conorii were applied to 93

the sorter and sorted with 1 sort event into 32 wells, 5 sort events into 32 wells, and 50 94

sort events into 32 wells of a 96 well plate containing Vero cells in complete DMEM 95

containing 200ng/mL Rifampicin. After 5 days of culture, the infected Vero cells were 96

lifted with Trypsin-EDTA and expanded into three identical 96 well plates containing 97

Vero cells in complete DMEM with 200ng/mL Rifampicin. Flow cytometric analyses and 98

cell sorting of pathogenic R. conorii were performed in a CDC-approved BSL-3 99

laboratory in the Department of Pathobiolgical Sciences at the LSU School of Veterinary 100

Medicine utilizing protocols approved by the LSU Institutional Biological and 101

Recombinant DNA Safety Committee (IBRDSC). 102

10 total days of growth after sorting, one of the three identical plates was fixed 103

with 4% paraformaldehyde. This plate was stained with the anti-R. conorii antibody 104

RcPFA (19) with anti-rabbit-AlexaFluor546 and observed under 10x magnification in an 105

Olympus IX71 inverted microscope for the presence of R. conorii (RcPFA) and GFPuv- 106

mediated fluorescence. Subsequently, DNA was extracted as described below from 107

wells containing R. conorii and demonstrating GFPuv-mediated fluorescence. Whole 108

DNA was extracted using the PureLink Genomic DNA kit (Life Technologies) according 109

to the manufacturer’s instructions. The extracted DNA was subjected to PCR using 110

gfpuv_F and gfpuv_R (Supplemental Table 1) followed by agarose gel electrophoresis 111

to ensure presence of pRam18dRGA[AmTrCh]. The clone utilized in this study came 112

from a single event sort into one well of a 96 well plate. 113

Immunofluorescence microscopy. R. conorii and R. conorii (pRam18dRGA[AmTrCh]) 114

were cultured in Vero cells. Both types of bacteria were purified by sucrose density 115


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gradient (17) and mounted onto a single glass slide using a Cytopro Dual sample 116

chamber with a Cytospin centrifuge. The bacteria were fixed with 4% paraformaldehyde 117

and stained using RcPFA with anti-rabbit-AlexaFluor350. All microscopy was captured 118

using a Leica DM4000B equipped with 100x oil objective, EvolutionOEi camera, with 119

ImagePro Plus software, using GFP (488/40nm ex 527/30nm em), TRITC (515-560nm 120

ex LP590nm em), and DAPI (360/40nm ex 470/40nm em) filters. Analysis of 121

fluorescent intensity was performed using the “RGB profile plot” plugin within the NIH 122

Image J freeware package (http://rsb.info.nih.gov/ij/). 123

Flow cytometric analysis. Sucrose purified bacteria were analyzed on a BD 124

FACSJazz equipped with aerosol containment and placed within a Baker class IIA 125

biosafety cabinet within the BSL-3 laboratory suite at the LSU School of Veterinary 126

Medicine. R. conorii (pRam18dRGA[AmTrCh]) were stained with anti-R. conorii 127

antibody RcPFA-PerCP/Cy5.5 at 10 µg/mL. All cytometric events were initially gated for 128

FSC and SSC to eliminate large events. PerCP/Cy5.5-mediated fluorescence was 129

measured at 488nm excitation and 692/40nm emission. GFPuv-mediated fluorescence 130

was measured at 488nm excitation and 530/40nm emission. This fluorescence 131

combination did not produce cross channel background so compensation was not 132

required. Each heat-map represents at least 50,000 FSC/SSC gated events. 133

Growth in culture. R. conorii and R. conorii (pRam18dRGA[AmTrCh]) were used to 134

infect Vero cells at a multiplicity of infection of 1 bacteria/ host cell. Bacteria were 135

allowed to grow at 34°C 5% CO2 for a total of 5 days in DMEM/10% FBS supplemented 136

with 200ng/mL Rifampicin for the transformed bacteria. At day = 1, 3, 5, 7, and 9 post-137


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infection, the infected Vero cells were scraped from the culture plate and DNA extracted 138

as above for host, R. conorii, and plasmid quantification. 139

R. conorii quantification. The chromosomal ratio of mouse or Vero (actin) to R. conorii 140

(sca1) was queried by quantitative PCR using TaqMan Master Mix at with a 95° 5 min 141

incubation followed by 55 cycles of 95°C 15sec, 53°C 15 sec, and 68°C 30 sec. Mouse 142

actin was amplified using actin_Ms_F, actin_Ms_R, and actin_Ms_Max(Vic). Vero actin 143

was amplified using actin_Vero_F, actin_Vero_R, ,and actin_V_Hex(Vic). R. conorii 144

sca1 was amplified using sca1_Rc_F, sca1_Rc_R, and sca1_Rc/Rr_Fam 145

(Supplemental Table 1). All unknowns were quantified by ∆∆Ct as compared to molar 146

standards. 147

Plasmid quantification. DNA from the above growth curve was used to quantify the 148

presence of the pRam18dRGA[AmTrCh] plasmid during growth in culture. The ratio of 149

plasmid (gfpuv) to R. conorii (sca1) was determined by quantitative PCR using SYBR 150

master mix (Roche) with a pre-incubation at 95°C for 5 min, followed by 50 cycles of 151

95°C 15sec, 55°C 15 sec, and 68°C 30 sec. Specificity of amplification was validated 152

by melting curve analysis. Gfpuv was amplified using gfpuv_F and gfpuv_R. R. conorii 153

sca1 was amplified using sca1_Rc_F and sca1_Rc_R. (Supplemental Table 1). 154

Animal infection. 5-7 week old male C3H/HeN mice were inoculated by retro-orbital 155

injection with 6x106 (1.5 LD50) R. conorii (n=5), R. conorii (pRam18dRGA[AmTrCh]) 156

(n=11) or PBS (n=5) (19). Infected mice were monitored twice daily for signs of disease 157

and daily for weight change. Animals that exhibited symptoms of severe disease that 158

were consistent with not recovering from the infection were removed from the study and 159

scored as succumbing to the infection as has been previously described (19). These 160


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symptoms included ruffled fur, hunched posture, shallow respiration, immobility when 161

touched, and weight loss of at least 15% of the initial body weight. Animal experiments 162

were conducted in accordance with protocols approved by the Institutional Biological 163

and Recombinant DNA Safety Committee (IBRDSC) and Institutional Animal Care and 164

Use Committee (IACUC) at the Louisiana State University School of Veterinary 165

Medicine. Two pre-designated mice were sacrificed at 1, 3, and 5 days post infection. 166

Spleens, kidneys, livers, hearts, lungs, brains, and blood were aseptically extracted to 167

determine the bacteria load, plasmid copy number, and assess pathological lesions. 168

Each organ was split into two sections for use in PCR analyses as described above and 169

pathological examination as described below. 170

Indirect Immunohistochemistry. Murine tissues, including lung, liver and spleen, 171

were collected immediately after euthanasia and fixed in 10% buffered formalin (1:10 172

tissue-formalin ratio). Samples were routinely processed and embedded in paraffin, 173

and 5-μm sections were cut for hematoxylin and eosin (H&E) staining. Isolated tissues 174

were additionally examined by immunohistochemistry to localize R. conorii within the 175

infected animals using anti-RcPFA, which recognizes several SFG rickettsial species 176

(19) or the macrophage-specific antigen, anti-Iba1. Primary antibody staining was 177

followed by biotinylated anti-rabbit IgG secondary antibody (1:1000, Vector BA 1000, 178

Vector Laboratories) and exposure to the detection reagent (Vectastain ABC Rabbit IgG 179

Kit, Vector PK 6102, Vector Laboratories). Slides were analyzed, micrographs 180

captured, and pathologic changes recorded by a Diplomate of the American College of 181

Veterinary Pathologists (DACVP). 182


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Identification of R. conorii in Leukocytes. Whole mouse blood was recovered by 183

cardiac puncture immediately after euthanasia. Blood was immediately anti-coagulated 184

using BD microtainer with K2EDTA, diluted 1:1 with PBS. Leukocytes were purified 185

using Ficoll-paque Premium (GE Healthcare) as directed by the manufacturer. 186

Leukocyte preparations were fixed with 4% paraformaldehyde, permeabilized with 0.1% 187

Triton X-100, and blocked with 2% Bovine Serum Albumin. For initial identification of R. 188

conorii infection, host nuclei were labeled with 1ng/mL DAPI and Phalloidin-Texas Red 189

(1:200, Life Sciences). These cells were visualized at 100x as described above. 190

Images were created by identification of fields containing GFPuv-positive bacilli, 191

followed by capture of Texas Red and DAPI images. Micrographs of the host cells 192

without the corresponding GFPuv-positive R. conorii image were scored for cell lineage 193

by a Diplomate of the American College of Veterinary Pathologists (DACVP). Only after 194

blinded identification of the host cell types, were the location and number of GFPuv-195

positive R. conorii attributed to each cell type. 196

Statistics. Bacterial growth in culture was analyzed by nonlinear fit analysis of the 197

slope and intercept. Plasmid content in culture and in mouse was analyzed by 1-way 198

ANOVA. Survival changes between R. conorii and R. conorii(pRam18dRGA[AmTrCh]) 199

were analyzed by comparison of survival curves via Log-rank (Mantel-Cox) test and 200

were analyzed from 5 mice per experimental group. All statistical analyses were 201

performed using the indicated tests within the Prism software package (GraphPad 202

Software Inc.).203


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RESULTS: 204

Transformation and isolation of pRam18dRGA[AmTrCh] transformed R. conorii in 205

cell culture. Previous reports had demonstrated the feasibility of using plasmids, 206

including pRam18dRGA[AmTrCh], to transform both recognized human pathogenic- 207

and non-pathogenic members of the genus Rickettsia (9-12, 15). Although successful, 208

these strategies have technical limitations in obtaining clonal transformed populations. 209

Use of either limiting dilution or plaque assays to isolate Rickettsia clones are time and 210

labor intensive, and repeated passage of these bacteria outside of a relevant animal 211

model can potentially contribute to attenuation (20). We sought to determine whether 212

transformation of R. conorii was achievable using this plasmid, and whether high-213

throughput fluorescence activated cell sorting (FACS) would be useful to isolate clonal 214

transformed R. conorii populations. 215

The plasmid pRam18dRGA[AmTrCh] (9) was introduced into purified R. conorii 216

Malish 7 by electroporation, and ,subsequently, transformed bacteria were subjected to 217

Rifampicin selection for a period of 10 days. After observing outgrowth of Rickettsia 218

bacilli via Diff-Quik staining and microscopic analysis, the infected host cells were lysed 219

by needle passage and unbroken cells were removed utilizing a 2µm filter. The 220

liberated bacteria were subsequently labeled with a PerCP/Cy5.5 conjugated anti-R. 221

conorii antibody (RcPFA, (19)) and subjected to flow cytometric analysis. Rifampicin-222

resistant fluorescent-labeled R. conorii were individually sorted using extremely 223

stringent single event sort using a FACS Jazz cell sorter (18). After outgrowth from the 224

single parent bacterium, clonal populations were screened for presence of GFPuv-225

positive R. conorii by PCR using primers specific for the gfpuv gene on the 226


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pRam18dRGA[AmTrCh] plasmid (data not shown). A single R. conorii 227

(pRam18dRGA[AmTrCh]) clone was propagated and purified by sucrose density 228

centrifugation for further analysis. 229

The pRam18dRGA[AmTrCh] plasmid encodes for two fluorescent proteins, 230

GFPuv and mCherry. mCherry expression is driven by the Anaplasma marginale 231

promoter tr, and the encoded protein fluoresces into the TRITC filter set (21, 22). gfpuv 232

expression is driven by the R. prowazekii ompA promoter, and GFPuv fluoresces 233

adequately into the FITC filter set (13). Accordingly, we hypothesized that transformed 234

R. conorii(pRam18dRGA[AmTrCh]) should readily fluoresce into the FITC (GFPuv) and 235

TRITC (mCherry) channels as has been previously observed in other Rickettsia species 236

(9, 15). As shown in Figure 1A, pRam18dRGA[AmTrCh] transformed R. conorii labeled 237

with the RcPFA antibody (19) are observable by fluorescence microscopy under TRITC 238

and FITC filter sets while untransformed bacteria are not visible. Analysis of the 239

fluorescence signal intensity across a portion of a R. conorii (pRam18dRGA[AmTrCh]) 240

photomicrograph indicates that individual bacterium elaborate both GFPuv and mCherry 241

signals (Figure 1B). 242

Flow cytometric analysis of GFPuv fluorescence. Since GFPuv can be excited 243

under a 488nm laser and emit into common filter sets (23), we hypothesized that 244

GFPuv-mediated fluorescence should be observable by flow cytometry in R. conorii 245

(pRam18dRGA[AmTrCh]). First, we conjugated the anti-R. conorii antibody RcPFA with 246

the PerCP/Cy5.5 tandem fluorophore. We used RcPFA-PerCP/Cy5.5 to identify R. 247

conorii when excited at 488nm and detected using 692/40nm emission filter. Indeed, 248

purified R. conorii are readily detectible by flow cytometry using RcPFA-PerCP/Cy5.5 249


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(Figure 2A vs. 2B). Additionally, GFPuv-mediated fluorescence from R. conorii 250

(pRam18dRGA[AmTrCh]) can be detected using FITC filter sets (488nm excitation and 251

530/40nm emission) (Figure 2A vs. 2C). As such, this is a novel method to identify 252

Rickettsia-specific events and to identify bacteria transformed to GFPuv fluorescence 253

(Figure 2D). 254

In vitro growth of transformed R. conorii in cell culture. 255

We next sought to determine if the presence of an extrachromosomal DNA element in 256

R. conorii has a deleterious affect on fitness in cell culture. R. conorii and R. conorii 257

(pRam18dRGA[AmTrCh]) were inoculated into Vero cell cultures at a MOI of 1. 258

Samples were removed from these cultures 1, 3, 5, 7, and 9 days post inoculation and 259

genomic DNA from both host and bacteria was extracted. Q-PCR analysis of the ratio 260

of R. conorii (sca1) to Vero (actin) DNA content indicates that transformed and non-261

transformed bacteria proliferate similarly over 9 days in Vero cell culture (Figure 3). 262

We then determined whether the plasmid was stably maintained in vitro without 263

selective antibiotic pressure. The copy number of pRam18dRGA[AmTrCh] was 264

determined by Q-PCR as the ratio of plasmid (gfpuv) to R. conorii chromosomal (sca1) 265

DNA. As demonstrated in Table 2, the pRam18dRGA[AmTrCh] copy number over the 266

time course of culture indicates that the plasmid is maintained without antibiotic 267

selection. The determined plasmid copy numbers over time are similar to those 268

documented in other transformed Rickettsia species (9, 11, 15). 269

R. conorii (pRam18dRGA[AmTrCh]) experimental mammalian infection. An 270

important question pertaining to the future of rickettsial genetics is the persistence of 271

extrachromosomal DNA in animal models of infection when lacking selective pressure. 272


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Indeed, pRam18dRGA can be maintained in R. monacensis for five passages in tissue 273

culture without antibiotic selection (9), but this phenotype has not been investigated in 274

an in vivo model. Additionally, future uses of extrachromosomal DNA will be dependent 275

on establishing that the existence of the plasmid does not, in itself, modulate the 276

infectivity of the bacterium. To address these questions, we utilized a well-established 277

murine model of fatal MSF to examine the pathogenesis of R. conorii 278

(pRam18dRGA[AmTrCh]) (19, 24). Susceptible C3H/HeN mice were intravenously 279

infected with 4x106 infectious units of R. conorii or R. conorii (pRam18dRGA[AmTrCh]) 280

This dose is 1.5 LD50 for non-transformed R. conorii (19). Mice were monitored for signs 281

of disease including: ruffled fur, hunched posture, shallow respiration, immobility, and 282

weight loss. R. conorii and R. conorii (pRam18dRGA[AmTrCh]) infected mice 283

demonstrated progressive morbidity as demonstrated by persistent weight loss (Figure 284

4A). As demonstrated in Figure 4B, infected mice succumbed to infection during the 285

normal course of disease with mortality at day 4-5.5 post-infection with a slight delay to 286

time of mortality as compared to WT. Nevertheless, this finding establishes that R. 287

conorii(pRam18dRGA[AmTrCh]) retain infectivity using a mouse model of infection. 288

To confirm that R. conorii (pRam18dRGA[AmTrCh]) infected mice followed the 289

normal disease progression, we quantified the bacterial burden in major organs. 290

Bacteria DNA could be detected and quantified in the mouse spleen and liver after 1 291

day of infection (Figure 5A). Total bacterial burden increased after 3 days of infection 292

(Figure 5B), and ultimately resulted in similar rickettsial organ tropism and quantity as 293

has been previously observed for R. conorii fatal infection (Figure 5C and (19)). Most 294

importantly, pRam18dRGA[AmTrCh] was readily detected at all time points and the 295


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plasmid quantity did not decrease over the course of infection (Figure 5D). Together, 296

these data demonstrate that the pRam18dRGA[AmTrCh] extrachromosomal DNA does 297

not affect R. conorii pathogenesis, and that the plasmid is stably maintained by R. 298

conorii in an animal model of infection. 299

Pathologic examination of animals after 5 days of infection demonstrated R. 300

conorii (pRam18dRGA[AmTrCh]) infection of endothelial cells in the heart and lung 301

(Supplemental Figure 1), as has been previously reported (24). The mouse liver 302

additionally demonstrated multifocal to coalescing coagulative hepatic necrosis 303

characterized by the presence of numerous neutrophils and leukocytic debris (Figure 304

6A). IHC staining of this lesion revealed the presence of numerous bacteria within the 305

cytoplasm of macrophages and neutrophils (Figure 6B). Additionally, bacterial were 306

noted to be infecting cells within vessels (Figure 6C). In the mouse liver, the 307

inflammatory cell population within vessels, sinusoids and microabscesses is mainly 308

comprised of round cells with macrophage morphology expressing the Iba1 309

macrophage marker (Figure 6D). There is no evidence of sloughed endothelial cells 310

within the lumen of blood vessels. These data demonstrate a normal distribution of 311

plasmid-transformed R. conorii in the infected animal along with a presence of these 312

bacteria within macrophages of in the liver. 313

Observation of ex vivo GFPuv-positive bacilli parasitizing leukocytes. We have 314

demonstrated that R. conorii (pRam18dRGA[AmTrCh]) is present within the 315

endothelium of the infected mammalian host (Supplemental Figure 1), but also within 316

several different cell types in infected target tissues and in circulation (Figure 6). In 317

addition, we demonstrated that the pRam18dRGA[AmTrCh] plasmid is stably 318


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maintained within infected cell cultures and in vivo (Figure 5D). We, therefore, 319

hypothesized that transformed R. conorii express genes encoded on the plasmid and 320

are readily detectable by endogenous GFPuv expression in vivo. To this end, we 321

sacrificed mice that were succumbing to the fatal R. conorii infection and isolated total 322

leukocytes from murine blood. After fixation and labeling of nucleus (DAPI) and host 323

cytoplasm (Phalloidin), GFPuv-fluorescent bacilli are readily observed within 324

granulocytic, monocytic, and lymphocytic cells (Figure 7). 325

We next sought to elucidate the different cell types present within the mammalian 326

bloodstream that are infected by R. conorii by determining the locations of the GFPuv-327

positive R. conorii within the ex vivo WBC preparation. GFPuv-positive bacilli were 328

located by fluorescence microscopy without observing location of the bacteria relative to 329

host cells. After detection of the bacteria, images of the host cells in that microscopic 330

field were acquired. A board-certified veterinary clinical pathologist identified the types 331

of leukocytes in the microscopic field without knowledge of the location of the bacteria. 332

Only after identification of host cells were the locations of the bacteria assigned. Of the 333

207 GFP-positive R. conorii that could be assigned to a host location, 110 were present 334

in lymphocytes, 67 were in monocytes, 14 were in granulocytic cells, and 16 were not 335

associated with any host cell (Table 2). Thus, these data suggest that in addition to 336

endothelial cells, cells of non-endothelial origin support R. conorii parasitism during fatal 337

mammalian infection.338


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DISCUSSION: 339

Herein, we have demonstrated the ability of R. conorii to be transformed with the 340

plasmid pRam18dRGA[AmTrCh] (9). R. conorii stably maintains this plasmid in culture 341

with no apparent affect on fitness, but with the addition of GFPuv- and mCherry-342

mediated fluorescence phenotypes (Figures 1, 2, 3, Table 1). By utilizing a well-343

characterized mouse model of fatal MSF, we were able to demonstrate that R. conorii 344

(pRam18dRGA[AmTrCh]) retains infectivity in a fatal mouse model of infection (Figure 345

4). Fatal R. conorii (pRam18dRGA[AmTrCh]) challenge results in disseminated 346

infection, and, importantly, retention of the extrachromosomal DNA (Figure 5). 347

Microscopic examination of R. conorii (pRam18dRGA[AmTrCh]) within the mouse 348

demonstrated the well-characterized endothelial-targeted infection (Supplemental 349

Figure 1), with newly-identified infection of mouse macrophages within the liver and 350

infection of non-endothelial cells within the microvascular circulation (Figure 6). Further 351

microscopic examination showed the presence of GFPuv-positive bacilli within 352

leukocytes (Figure 7), and pathologic examination was applied to identify the nature of 353

this host infection (Table 2). Taken together, these data indicate that use of 354

pRam18dRGA plasmids will add depth to our analysis of mammalian models of 355

Rickettsia infection. The GFPuv-mediated fluorescence phenotype will permit a more 356

complete analysis of the nature of the pathogenic process and to identify host cells 357

parasitized by pathogenic Rickettsia species. 358

In vivo maintenance of pRam18dRGA plasmids (Figures 5 and 7) and the 359

adaptable nature of the pRam18dRGA[MCS] plasmid allows additional approaches to 360

examining Rickettsia pathogenesis. The pRam18dRGA[MCS] plasmid contains a 361


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multiple cloning site for insertion of any DNA (9). This design feature makes this 362

plasmid a true shuttle vector, because the additional DNA will be maintained within the 363

bacteria during in vivo Rickettsia infection. First and foremost among potential uses of 364

shuttle vectors is proper and complete examination of gene deletion mutants, because 365

of the essential need for in trans gene complementation (25). pRam18dRGA[MCS] 366

derivatives can also be utilized to monitor bacterial gene expression through the 367

insertion of appropriate reporter genes, analysis of natural conjugation systems, or for 368

heterologous expression of many different bacterial genes (9, 26). 369

Of particular importance is our ex vivo microscopic observance of fluorescent R. 370

conorii directly from the infected animal. Analysis of in vivo or ex vivo fluorescent bacilli 371

will promote a more complete examination of Rickettsia pathogenesis, as has been 372

done in other bacteria (27). This microscopic technique can also be readily applied to: 373

examine cell types infected parasitized by pathogenic Rickettsia, observe arthropod 374

infection in real time, and follow the kinetics of rickettsemia in infected mammals. 375

SFG Rickettsia have long been known to infect the endothelium of the infected 376

animal (24, 28, 29). In this manuscript, we provide evidence of non-endothelial 377

parasitism by R. conorii (Figures 6 and 7, Table 2). We propose that infection of non-378

endothelial cells has been overlooked during prior examination of SFG Rickettsia 379

pathogenesis, and that our newly developed techniques have allowed a more complete 380

analysis of infection. This idea is supported by several in vitro and ex vivo studies 381

which have demonstrated that rickettsial species can infect and grow within 382

macrophages and hepatocytes (30-33), suggesting that the interaction with cells other 383

than the endothelium may be an important and underappreciated aspect in the 384


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pathogenesis of rickettsial diseases. Additionally, immunohistochemical and 385

immunofluorescence microscopy both display intact bacilli, and non-endothelial cells 386

frequently contain multiple plasmid-transformed R. conorii (Figures 6 and 7), suggesting 387

that these bacteria may not be readily susceptible to immune-mediated killing. A more 388

complete examination of the viability of bacilli, infection kinetics, and host tropism of R. 389

conorii parasitism is necessary. 390

In conclusion, we have utilized R. conorii (pRam18dRGA[AmTrCh]) to 391

demonstrate the value of plasmids in analysis of in vivo Rickettsia pathogenesis . The 392

techniques described herein have promoted our identification of novel host cell types 393

parasitized by pathogenic R. conorii in a fatal model of Mediterranean spotted fever. 394

Therefore, the use of pRam18dRGA[AmTrCh] and related plasmids will strongly 395

influence and enhance the course of future rickettsial research.396


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FUNDING INFORMATION: 397

These studies were funded in part by NIH-NIAID award R01AI1072606 to J.J.M. and 398

NIH-NIGMS award P30GM110760 Pilot Grant to S.P.R. The content is solely the 399

responsibility of the authors and does not necessarily represent the official views of the 400

National Institutes of Health.401


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ACKNOWLEDGEMENTS: 402

The authors would like to thank Dr. Kevin Macaluso (LSU-SVM) for helpful comments 403

on experimental design and manuscript construction, Dr. David Wood (University of 404

South Alabama) for dissemination of techniques for electrotransformation, Dr. Ulrike 405

Munderloh (University of Minnesota) for distribution of reagents, and Dr. Rebecca 406

Christofferson (LSU-SVM) for aid in the statistical analysis of data.407


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9. Burkhardt NY, Baldridge GD, Williamson PC, Billingsley PM, Heu CC, 432

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10. Baldridge GD, Burkhardt NY, Felsheim RF, Kurtti TJ, Munderloh UG. 2008. 435

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Williamson PC, Billingsley PM, Felsheim RF, Kurtti TJ, Munderloh UG. 2010. 439

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Microbiol 76:1718-1731. 442

12. Baldridge GD, Burkhardt NY, Oliva AS, Kurtti TJ, Munderloh UG. 2010. 443

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13. Liu ZM, Tucker AM, Driskell LO, Wood DO. 2007. Mariner-based transposon 446

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animals with Rickettsia prowazekii of reduced pathogenicity, strain E. Am J Hyg 464

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RY. 2004. Improved monomeric red, orange and yellow fluorescent proteins 471

derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 22:1567-472

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368. 479

25. Falkow S. 1988. Molecular Koch's postulates applied to microbial pathogenicity. 480

Rev Infect Dis 10 Suppl 2:S274-276. 481

26. Lee SH, Camilli A. 2000. Novel approaches to monitor bacterial gene 482

expression in infected tissue and host. Curr Opin Microbiol 3:97-101. 483

27. Konjufca V, Miller MJ. 2009. Imaging Listeria monocytogenes infection in vivo. 484

Curr Top Microbiol Immunol 334:199-226. 485

28. Feng HM, Wen J, Walker DH. 1993. Rickettsia australis infection: a murine 486

model of a highly invasive vasculopathic rickettsiosis. Am J Pathol 142:1471-487

1482. 488

29. Walker DH, Crawford CG, Cain BG. 1980. Rickettsial infection of the pulmonary 489

microcirculation: the basis for interstitial pneumonitis in Rocky Mountain spotted 490

fever. Hum Pathol 11:263-272. 491

30. Walker DH, Popov VL, Feng HM. 2000. Establishment of a novel endothelial 492

target mouse model of a typhus group rickettsiosis: evidence for critical roles for 493

gamma interferon and CD8 T lymphocytes. Lab Invest 80:1361-1372. 494


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31. Gambrill MR, Wisseman CL, Jr. 1973. Mechanisms of immunity in typhus 495

infections. 3. Influence of human immune serum and complement on the fate of 496

Rickettsia mooseri within the human macrophages. Infect Immun 8:631-640. 497

32. Oster CN, Burke DS, Kenyon RH, Ascher MS, Harber P, Pedersen CE, Jr. 498

1977. Laboratory-acquired Rocky Mountain spotted fever. The hazard of aerosol 499

transmission. N Engl J Med 297:859-863. 500

33. Radulovic S, Price PW, Beier MS, Gaywee J, Macaluso JA, Azad A. 2002. 501

Rickettsia-macrophage interactions: host cell responses to Rickettsia akari and 502

Rickettsia typhi. Infect Immun 70:2576-2582. 503

504

505


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TABLES: 506

Table 1. Plasmid copy number during Vero cell growth. 507

Days of culture Plasmid copy number (Average ± Standard Deviation)*

3 3.09 ± 0.52

5 2.82 ± 2.03

7 0.52 ± 0.30

9 0.55 ± 0.14

*Changes in copy number are not significant. 508

509

Table 2. Analysis of the cellular location of GFPuv-positive bacilli in an ex vivo 510

leukocyte preparation. 511

Total number of bacteria counted 207

Bacteria present in lymphocytes 110

Bacteria in monocytes 67

Bacteria in granulocytes 14

Bacteria not associated with a host cell 16

512

513


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FIGURE LEGENDS: 514

Figure 1. in vitro fluorescent properties of R. conorii (pRam18dRGA[AmTrCh]). 515

A. R. conorii and R. conorii (pRam18dRGA[AmTrCh]) were purified and stained with the 516

anti-Rickettsia antibody RcPFA with corresponding anti-rabbit AlexaFluor350 (blue). 517

Surface staining of the bacteria is apparent in both anti-RcPFA panels, but only R. 518

conorii (pRam18dRGA[AmTrCh]) demonstrate GFPuv- (green) and mCherry- (red) 519

mediated fluorescence. B. Unstained R. conorii (pRam18dRGA[AmTrCh]) were 520

analyzed for GFPuv- and mCherry- mediated fluorescence. Analysis of fluorescence 521

intensity along the magenta line indicates that individual bacteria elaborate both GFPuv- 522

and mCherry- mediated fluorescence as demonstrated by overlapping peaks. However, 523

the mCherry fluorescence is not always above the limit of detection. 524

525

Figure 2. Flow cytometric analysis of GFPuv- mediated fluorescence. A. Non-526

labeled R. conorii, B. RcPFA-PerCP/Cy5.5- labeled R. conorii, C. Non-labeled R. conorii 527

(pRam18dRGA[AmTrCh]) and, D. RcPFA-PerCP/Cy5.-labeled R. conorii 528

(pRam18dRGA[AmTrCh]) were analyzed by flow cytometry for identification of R. 529

conorii specific events (RcPFA) and GFPuv fluorescence. RcPFA-PerCP/Cy5.5 labels 530

both R. conorii (A vs. B) and R. conorii (pRam18dRGA[AmTrCh]) (C vs. D). R. conorii 531

(pRam18dRGA[AmTrCh]) demonstrates significant intrinsic GFPuv-mediated 532

fluorescence (A vs. C and B vs. D). 533

534

Figure 3. Presence of pRam18dRGA[AmTrCh] does not alter R. conorii fitness in 535

culture. R. conorii (pRam18dRGA[AmTrCh]) (dashed line) and plasmid-free R. conorii 536


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(solid line) proliferate similarly in Vero cell culture. Quantitative PCR data are 537

expressed as the ratio of R. conorii sca1 versus Vero actin DNA after 3, 5, 7, and 9 538

days of culture in Vero cells. Differences in slope or intercept are not significant. 539

540

Figure 4. R. conorii (pRam18dRGA[AmTrCh]) retains virulence in a fatal mouse 541

model of infection. A. Mice were infected with R. conorii (dashed line), R. conorii 542

(pRam18dRGA[AmTrCh]) (dotted line) or PBS (solid line) and were monitored for 543

morbidity as determined by weight change over the course of infection. B. The same 544

infected mice were monitored for survival. R. conorii harboring pRam18dRGA[AmTrCh] 545

are capable of producing fatal disease. Time to euthanasia p<0.05. 546

547

Figure 5. R. conorii(pRam18dRGA[AmTrCh]) proliferates within infected organs 548

and maintains extrachromosomal plasmid. R. conorii (pRam18dRGA[AmTrCh]) 549

dissemination into mouse organs after 1 (A.), 3 (B.), and 5 (C.) days of infection. 550

Chromosomal equivalents of R. conorii sca1 and mouse actin were determined by 551

quantitative PCR. D. Non-selective persistence of pRam18dRGA[AmTrCh] throughout 552

the mammalian infection as determined by quantitative PCR ratio of chromosomal R. 553

conorii sca1 and the plasmid gene gfpuv. Change in plasmid copy number is not 554

significant. 555

556

Figure 6. Pathological examination of mouse liver. A. Hematoxylin and eosin stain 557

of mouse liver demonstrates microabscess development when animal is succumbing to 558

infection. B. Immunohistochemical staining using anti-R. conorii antibody with 559


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hematoxylin counter stain demonstrates presence of Rickettsia antigen-positive intact 560

bacilli (brown) within liver microabscess. C. Rickettsia antigen- positive intact bacilli 561

(brown) within circulating cells of liver capillary. D. Iba1- positive macrophages (brown) 562

within both a liver venule (lower right) and microabscesses (arrow heads). All images 563

were captured at 60x. 564

565

Figure 7. ex vivo fluorescence of R. conorii (pRam18dRGA[AmTrCh]). Microscopic 566

identification of R. conorii (pRam18dRGA[AmTrCh]) infected cells from a white blood 567

cell preparation. Cells are stained with DAPI (blue) to identify host nuclei and Phalloidin 568

(red) to illuminate the cytoplasm. Green fluorescence results from intrinsic GFPuv-569

mediated fluorescence as encoded by pRam18dRGA[AmTrCh]. Infected host cells 570

demonstrate intact GFPuv-positive bacilli. White bars = 10μm. All images were 571

captured at 100x. 572


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